Preparation of ganglioside having ceramide moiety labeled with fluorescence

ABSTRACT

A ganglioside derivative in which a ceramide moiety is labeled with a fluorescent dye represented by the formula (I): ##STR1## wherein --O--R--O-- is a saccharide chain constituting the ganglioside, R&#39; is an ether, sulfide, amide, urethane, thiourea or amino group having a fluorescent dye, one of R 5  or R 6  is a single bond and the other is a hydrogen atom, I is an integer of 1 to 8, m is an integer of at least 2, and n is an integer of 0 to 12. This ganglioside derivative has recognition characteristics of the saccharide chain portion and the labeling property of the fluorescent dye introduced in the ceramide moiety, and is useful as an agent for monitoring the dynamic behavior of the receptor for glycolipids, a diagnostic agent for bacteria or viruses, and the like.

This is the U.S. national stage entry under 35 U.S.C. 371 ofPCT/JP95/00951, filed May 18, 1995.

FIELD OF THE INVENTION

The present invention relates to a ganglioside having a fluorescentlylabeled ceramide moiety, intermediates for the synthesis thereof, and aprocess for the preparation thereof.

PRIOR ART

Glycolipids in mammalian cells belong to the group of sphingoglycolipidscomprising a lipid portion called as a ceramide which consists of asphingosine as a long chain aminoalcohol and a long chain fatty acidbonded to the sphingosine through an amide linkage, variousoligosaccharide chains and a sialic acid. In particular, ganglioside isthe collective name for sphingoglycolipids having a sialic acid group.

In general, most of the above ganglioside molecules are localized in thecell surfaces of animals, and the recent studies have revealed thattheir sialic saccharide chains are oriented outside the cells and thatthey play important roles in fundamental life phenomena such asdiscrimination in cells, reception of or response to information incells, functions as receptors for hormones, viruses, bacteria, celltoxins, etc., intercellular distinction, proliferation and metastasis ofcells, malignant alteration, immunity, and the like.

The gangliosides attract attentions as the molecules which exhibit awide variety of physiological activities which relate to the informationtransfer, proliferation and metastasis of the cells.

A derivative of the ganglioside into which a fluorescent dye isintroduced while maintaining its inherent physiological activities willprovide a novel method for the study of cells, if such a derivative canbe synthesized, since the ganglioside receptor can be identified and itsbehavior can be monitored visually.

A ganglioside into which fluorescein is introduced has been preparedbased on the above idea. However, it has not achieved satisfactoryresults.

A method for detecting a bond between a ganglioside and bacteria orviruses using the ganglioside labeled with a radioisotope has beendeveloped. However, a new method alternative to this method has beensought since this method has various problems in handling, and labelingwith a fluorescence is thought to be effective.

A ganglioside labeled with a fluorescent dye will be useful as adetection and diagnostic agent for the above pathogens.

SUMMARY OF THE INVENTION

The first object of the present invention is to provide a gangliosidederivative into which a labeling dye has been introduced withoutdeterioration of the physiological activities of the ganglioside.

The second object of the present invention is to provide an intermediatefor the synthesis of such a ganglioside derivative.

The third object of the present invention is to provide processes forthe preparation of such ganglioside derivative and the intermediate forthe same.

According to the first aspect of the present invention, a gangliosidederivative labeled with a fluorescent dye is provided, represented bythe formula (I): ##STR2## wherein --O--R--O-- is a saccharide chainconstituting the ganglioside, R' is an ether, sulfide, amide, urethane,thiourea or amino group having a fluorescent dye, one of R⁵ or R⁶ is asingle bond and the other is a hydrogen atom, I is an integer of 1 to 8,m is an integer of at least 2, and n is an integer of 0 to 12.

According to the second aspect of the present invention, a gangliosidederivative labeled with a fluorescent dye is provided, represented bythe formula (II): ##STR3## wherein R^(2') is a protecting group for acarboxylic acid, R^(3'), R^(7'), R^(8') and R^(9') are protecting groupsfor hydroxyl groups, one of R^(5') or R^(6') is a single bond and theother is a hydrogen atom, and --O--R--O--, R', I, m and n are the sameas defined above.

DETAILED DESCRIPTION OF THE INVENTION

The ganglioside of the present invention is characterized in that thefluorescent dye as a label is introduced in the ceramide moiety of theganglioside.

The kind of fluorescent dye is not limited. Preferable examples of thefluorescent dye are fluorescein, 7-hydroxycoumarin, 7-aminocoumarin,2,4-dinitrophenyl, pyrene, anthracene, acridine, cascade blue,rhodamine, 4-benzoylphenyl, Rosamine, 7-nitrobenz-2-oxa-1,3-diazole,4,4-difluoro-4-bora-3a,4a-diaza-3-indacene, 5-azidonaphthalene, and thelike. These fluorescent dyes may have substituents.

The fluorescent dye is amide bonded to the ceramide moiety through theether, sulfide, amide, urethane, thiourea or amino group and also amethylene chain. The number of carbon atoms in the methylene chaininterposed between the amide linkage and the labeling dye is between 2and 20, preferably between 5 and 10.

Examples of the saccharide chain constituting the ganglioside accordingto the present invention, that is, --O--R--O-- in the formulas (I) and(II) are

β-D-O-galactopyranosyl-(1→4)-β-D-glucopyranosyl,

0-(2-acetamide-2-deoxy-β-D-galactopyranosyl)-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl,

0-β-D-galactopyranosyl-(1→3)-O-(2-acetamide-2-deoxy-β-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl,

O-β-D-galactopyranosyl-(1→3)-O-(2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl,

O-β-D-galactopyranosyl-(1→4)-O-(2-acetamide-2-deoxy-β-D-glucopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-β-D-glucopyranosyl,

O-(2-acetamide-2-deoxy-β-D-galactopyranosyl)-(1→4)-O-α-D-galactopyranosyl-(1→4)-O-β-D-galactopyranosyl-(1→4)-.beta.-D-glucopyranosyl,

O-(2-acetamide-2-deoxy-β-D-galactopyranosyl)-(1→4)-O-α-D-galactopyranosyl-(1→3)-O-β-D-galactopyranosyl-(1→4)-.beta.-D-glucopyranosyl,

O-β-D-galactopyranosyl-(1→4)-O-(O-α-L-fucosyl-(1-3))-(2-acetamide-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranosyl,or

O-β-D-galactopyranosyl-(1→3)-O-(O-α-L-fucosyl-(1-4))-(2-acetamide-2-deoxy-β-D-glucopyranosyl)-(1→3)-O-β-D-galactopyranosyl-(1→4)-O-β-D-glucopyranosyl

The compound of the formula (I) according to the present invention maybe synthesized by the following reaction steps:

First, a compound of the formula (VII): ##STR4## wherein --O--R--O-- isa saccharide chain constituting the ganglioside, one of R^(5') or R^(6')is a single bond and the other is a hydrogen atom, R^(3'), R^(7'),R^(8') and R^(9') are protecting groups for hydroxyl groups, R^(2') is aprotecting group for a carboxylic acid, and n is an integer of 0 to 12is condensed with a compound of the formula (VII):

    HOOC--(CH.sub.2).sub.m --R'                                (VIII)

wherein m is an integer of at least 2, and R' is an ether, sulfide,amide, urethane, thiourea or amino group having a fluorescent dye toobtain a ganglioside of the formula (II): ##STR5## wherein --O--R--O--,R', R^(2'), R^(3'), R^(5'), R^(6'), R^(7'), R^(8'), R^(9'), I, m and nare the same as defined above.

Examples of the protecting group for the hydroxyl group in the compoundof the formula (VII) are ester groups such as an acetyl, benzoyl andpivaloyl groups, and examples of the protecting group for the carboxylicacid are lower alkyl groups such as a methyl, ethyl, propyl and butylgroups, and a benzyl group.

The compound of the formula (VII) may be prepared as follows:

The saccharide chain portion (--O--R--O--) is synthesized by protectinghydroxyl groups of at least one monosaccharide selected from the groupconsisting of D-glucose, D-galactose, N-acetyl-D-glucosamine,N-acetyl-D-galactosamine, L-fucose, lactose and N-acetyllactosamine, andcondensing the protected monosaccharide by the glycosidation method(see, for example, the Journal of the Organic Synthesis Society (YUKIGOSEI KYOKAI-SHI), Vol. 60 (1992) 378-400).

The obtained saccharide chain is condensed with methyl(methyl-5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-.alpha.-D-galacto-2-nonulopiranosid)onatein the presence of an activating agent such asN-iodo-succinimide/trifluoromethane-sulfonic acid or dimethylmethylthiosulfonium triflate. When I is 2 or larger, the obtained sialicacid to which the saccharide chain has been bonded is condensed with theabove compound in the same manner as above to add the sialic acidsuccessively. Then, the sialylated saccharide chain is deprotected atthe reducing terminal, and a sialylated saccharide chain of the formula:##STR6## wherein --O--R--O--, R^(2'), R^(5'), R^(6'), R^(7'), R^(8'),R^(9') and I are the same as defined above, is obtained.

The hydroxyl group at the saccharide chain end of this compound isactivated with an activatable substituent, for example, bytrichloroacetimidation, and then condensed with an azidosphingosine ofthe formula: ##STR7## wherein R³ and m are the same as defined above,which has been synthesized by the method described by K. C. Nicolaus inCarbohydr. Res., Vol. 202 (1990) 177-191, and an azide compound of theformula (X): ##STR8## wherein R^(2'), R^(3'), R^(5'), R^(6'), R^(7'),R^(8'), R^(9'), I and n are the same as defined above, is obtained.

Then, the azido group of this compound is converted to an amino groupwith a triphenylphosphine/water system or a hydrogen sulfide/pyridinesystem, and the compound of the formula (VII) is obtained. The exampleof this synthesis is described in JP-A-3-101691, page 4, lower rightcolumn, line 7 to page 9, upper right column, line 3.

The compound of the formula (VIII) may be prepared as follows:

First, an iodomethyl (--CH₂ I), isocyanate (--NCO), thioisocyanate(--NCS), amino (--NH₂) or carboxyl (--COOH) group is introduced into thefluorescent dye such as fluorescein, 7-hydroxycoumarin, 7-aminocoumarin,2,4-dinitrophenyl, pyrene, anthracene, acridine, cascade blue,rhodamine, 4-benzoylphenyl, Rosamine, 7-nitrobenz-2-oxa-1,3-diazole,4,4-difluoro-4-bora-3a,4a-diaza-3-indacene, 5-azidonaphthalene, and thelike. The methods for introducing such groups are known, and compoundsinto having such groups are commercially sold and easily obtained.

Then, the above obtained compound is bonded to a dicarboxylic acid (a),ω-amino acid (b), ω-hydroxycarboxylic acid (c) or ω-thiolcarboxylic acid(d) of the formula:

    HOOC(CH.sub.2).sub.m COOH                                  (a)

    HOOC(CH.sub.2).sub.m NH.sub.2                              (b)

    HOOC(CH.sub.2).sub.m OH                                    (c)

    or HOOC(CH.sub.2).sub.m SH                                 (d)

wherein m is an integer of at least 2.

The fluorescent dye having the iodomethyl group reacts with the aminogroup of the amino acid (b), the OH group of the hydroxycarboxylic acid(c) or the thiol group of the thiolcarboxylic acid (d) to form the aminobond, ether bond or sulfide bond, respectively. The fluorescent dyehaving the thioisocyanate group reacts with the amino group of the aminoacid (b) to form the thiourea bond. The fluorescent dye having the aminogroup is condensed with the carboxyl group of the dicarboxylic acid (a)in the presence of a dehydrating agent such as dicyclohexylcarbodiimideto form the amide group. The fluorescent dye having the carboxyl groupis condensed with the amino acid (b) to form the amide bond.

Accordingly, a compound of the formula (VIII):

    HOOC--(CH.sub.2).sub.m --R'                                (VIII)

wherein m is an integer of at least 2, and R' is an ether, sulfide,amide, urethane, thiourea or amino group having a fluorescent dye isobtained.

The above reactions can be performed according to the conventionalmethods in the organic syntheses.

Next, the amino group of the compound of the formula (VII) is condensedwith the carboxyl group of the compound of the formula (VIII) in thepresence of a dehydrating agent such as dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIPC),N-ethyl-N'-3-dimethylaminopropylcarbodiimide (WSCI), and the like, andthe compound of the formula (II) is obtained.

The molar ratio of the compound of the formula (VII) to the compound ofthe formula (VIII) in the above reaction is between 1:0.5 and 1:2.0,preferably between 1:1 and 1:1.1.

The dehydrating agent is used in an amount of between 1 and 2 moles,preferably between 1 and 1.1 moles per one mole of the compound of theformula (VII).

Examples of preferably used solvents are dichloromethane, chloroform,dichloroethane, dimethylformamide, and the like.

The reaction temperature is usually between 15° and 25° C.

After the completion of the reaction, the reaction mixture ispost-treated by, for example, extraction or evaporation of the solvent.The product may be purified by column chromatography, if necessary.

Alternatively, the compound of the formula (II) may be prepared byactivating the carboxyl group of the compound of the formula (VIII) andthen reacting the activated carboxyl group with the amino group of thecompound of the formula (VII) according to the conventional method forthe peptide synthesis, instead of the direct condensation between thecompound of the formula (VII) and the compound of the formula (VIII) inthe presence of the dehydrating agent such as DCC. The carboxyl groupcan be activated by the conventional methods such as the formation of anactive ester with N-hydroxysuccinimide or p-nitrophenol, the mixed acidanhydride method, and the like.

The method using N-hydroxysuccinimide will be explained by way ofexample.

The compound of the formula (VII) is reacted with N-hydroxysuccinimidein the presence of a dehydrating agent such as DCC to obtain aN-hydroxysuccinimide ester of the formula (IX): ##STR9## wherein m isthe same as defined above.

This compound of the formula (IX) and the compound of the formula (VII)are condensed to obtain the compound of the formula (II). The compoundof the formula (IX) is used in an amount of between 1 to 10 moles perone mole of the compound of the formula (VII). No catalyst may benecessary in general, although organic bases such as pyridine,triethylamine, etc. or inorganic bases such as sodium carbonate,potassium carbonate, etc. may be used as catalysts.

Aprotic solvents such as dichloromethane, dichloroethane, chloroform,ethyl acetate, dimethylformamide, and the like can be used as reactionmedia. The reaction temperature is between 0° and 40° C.

Then, the protecting groups for the hydroxyl and carboxyl groups of thecompound of the formula (II) are removed, and the ganglioside (I)labeled with the fluorescence according to the present invention isobtained.

The above deprotection reaction can be performed as follows:

The compound of the formula (II) is dissolved in anhydrous methanol, andthe two to four times molar equivalents of sodium methoxide is added tothe solution. Then, the mixture is reacted at 50° C. for 30 minutes to10 hours to remove the protecting groups for the hydroxyl and carboxylgroups. The reaction mixture is cooled to 0° C., and water is added tothe mixture and stirred for 1 to 6 hours at the same temperature. Themixture is desalted with a H type cation exchange resin and thenpurified with a column containing SEPHADEX LH-20 to obtain theganglioside (I) labeled with the fluorescent dye.

The preparation of fluorescein ganglioside GM3 as one of the gangliosidelabeled with the fluorescent dye according to the present invention willbe explained.

First, a compound of the formula (III): ##STR10## wherein n is aninteger of 0 to 12, R¹, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R³ areprotecting groups for the hydroxyl groups, and R² is a protecting groupfor the carboxyl group, is reacted with a compound of the formula (IV):##STR11## wherein m is an integer of at least 2, and R⁴ is a protectinggroup for the hydroxyl group, and the compound of the formula (V):##STR12## wherein R¹, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R³, R⁴, m and n arethe same as defined above.

The compound of the formula (IV) can be prepared as follows:

5-(or -6-)carboxyfluorescein and methyl ω-aminohexanoate hydrochlorideare reacted in dimethylformamide in the presence of WSCI and pyridine,and 6-(fluorescein)-5 (or -6-)carboxamidehexanoic acid is obtained. Thismethyl ester is hydrolyzed with an alkali and then condensed withN-hydroxysuccinimide in the presence of WSCI as the carboxyl groupsource, and the compound of the formula (IV) is obtained.

Then, the protecting groups for the hydroxyl and carboxyl groups of thecompound (V) are removed, and the fluorescein ganglioside GM3 of thefollowing formula (VI) is obtained: ##STR13## wherein m and n are thesame as defined above.

EXAMPLES

The present invention will be illustrated by the following examples,which do not limit the scope of the present invention.

The abbreviations used in the results of NMR are as follows:

Me: Methyl group

Ac: Acetyl group

Ph: Phenyl group.

A ganglioside labeled with a fluorescent dye and an intermediate for thesame of the present invention were prepared according to the followingreaction scheme: ##STR14##

Example 1

Synthesis of O-(methyl5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonate)-(2→3)-O-(2,4-di-O-acetyl-6-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-(3-O-acetyl-2,6-di-O-benzoyl-β-D-glucopyroanosyl)(1→1)-(2S,3R,4E)-3-O-benzoyl-2- 6-{fluorescein-5- (and-6-)-carboxamide}hexanamide!-4-octadecen-1,3-diol (the compound 3 in theabove reaction scheme, hereinafter, referred to as the compound (3))

O-(Methyl5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonic)-(2→3)-O-(2,4-di-O-acetyl-6-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-(3-O-acetyl-2,6-di-O-benzoyl-β-D-glucopyranosyl)(1→1)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol(the compound 1 in the above reaction scheme) (60 mg, 0.036 mmol) wasdissolved in a mixed solvent of pyridine (5 ml) and water (1 ml).Hydrogen sulfide gas was bubbled through the solution at roomtemperature for 55 hours to reduce the azide group of the compound (1)to an amino group. After confirming the consumption of the raw material,hydrogen sulfide was removed from the reaction mixture, and thenpyridine and water were evaporated off in vacuo.

The residue was dissolved in a mixed solvent of anhydrousdichloromethane (2 ml) and anhydrous N,N-dimethylformamide (1 ml), andsuccinimidyl 6-(fluorescein-5- (and -6-)-carboxamide)hexanoate(hereinafter referred to as SFX) (30 mg, 0.051 mmol) was added to thesolution in an argon atmosphere, followed by stirring at roomtemperature for 18 hours. Further, SFX (47 ml, 0.080 mmol) was added,and the mixture was stirred at room temperature for 18 hours.

The reaction mixture was diluted with ethyl acetate, washed with water,dried over anhydrous sodium sulfate, and concentrated under reducedpressure. The residue was subjected to silica gel column chromatography(packing: silica gel 60 (7734), eluent: methanol/chloroform=1/15→1/10),and the compound (3) (35 mg) was obtained. Yield: 46%.

IR_(max) ^(KBr) (cm⁻¹): 3430 (NH, OH), 2930, 2860 (Me, methylene) 1745,1230 (ester), 1650, 1370 (amide), 710 (phenyl). NMR (CDCl₃ ; TMS):

Lactose unit: δ 4.59 (dd, J=10 Hz, H-3'), 4.67 (d, J=7.5 Hz, 1H, H-1),4.83 (d, J=8 Hz, 1H, H-1'), 5.00 (d, 1H, J=4 Hz, H-4'), 5.01 (dd, J=10Hz, 8 Hz, 1H, H-2'), 7.3-8.1 (m, 20H, 4×Ph).

Sialic acid unit: δ 1.66 (dd, J=13 Hz, 13 Hz, 1H, H-3α), 1.84 (s, 3H,N--COCH₃), 2.58 (dd, J=13 Hz, 5 Hz, 1H, H-3e), 3.70 (s, 3H, OCH₃), 4.86(m, 1H, H-4).

Ceramide unit: δ 0.86 (t, J=7 Hz, 3H, CH₃), 1.3-1.1 (m, 26H, 13×CH₂),5.50 (dd, J=15 Hz, 7 Hz, 1H, H-4), 5.67 (dt, J=15 Hz, 7 Hz, 1 H, H-5).

O-Acetyl group: δ 1.965, 1.975, 1.99(2), 2.01, 2.10, 2.18 (7s, 21H,7×Ac).

Example 2

Synthesis ofO-(5-acetamide-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosilonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-β-D-glucopyranosyl)(1→1)-(2S,3R,4E)-2-6-{fluorescein-5-(and -6-) -carboxamide}hexanamide!-4-octadecen-1,3-diol(the compound 4 in the above reaction scheme, hereinafter referred to asthe compound (4)).

Sodium methoxide (10 mg, 0.185 mmol) was added to a solution of thecompound (3) (34 mg, 0.016 mmol) in anhydrous methanol (2 ml) in anargon atmosphere and stirred at room temperature for 8 hours. Aftercooling to 0° C., water (0.2 ml) was added to the reaction mixture andstirred at 0° C. for 5.5 hours. The reaction mixture was subjected tothe AMBERLITE IR 120 (H⁺) column chromatography (eluent:methanol/water=9/1), and the eluted portion was evaporated under reducedpressure. The residue was subjected to column chromatography (packing:SEPHADEX LH-20, eluent: methanol), and the compound (4) (19 mg) wasobtained.

Yield: 85%.

α!_(D) ²⁵ =0.49° (c=0.21, 1:2 CHCl₃ /CH₃ OH). IR_(max) ^(KBr) (cm⁻¹):3380 (OH, NH), 2930, 2855 (Me, methylene), 1745 (carbonyl) 1635, 1550(amide). NMR (3:1 CD₃ OD/CDCl₃, TMS):

Lactose unit: δ 4.30 (d, J=8 Hz, 1H, H-1), 4.42 (d, J=8 Hz, 1H, H-1').

Sialic acid unit: δ 2.02 (s, 3H, N-COCH₃), 2.76 (dd, J=13 Hz, 5 Hz, 1 H,H-3e).

Ceramide unit: δ 0.88 (t, J=7 Hz, 3H, CH₂ CH₃), 1.3-1.1 (m, 26H,13×CH₂), 2.24 (t, J=7 Hz, 2H, CH₂ CO), 4.17 (dd, J=10 Hz, 5 Hz, 1H,H-1), 5.46 (dd, J=15 Hz, 7 Hz, 1H, H-4), 5.71 (dt, J=15 Hz, 1H, H-5).##STR15##

Example 3

Synthesis of O-(methyl5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyrasylonate)-(2→3)-O-(2,4-di-O-acetyl-6-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-(3-O-acetyl-2,6-di-O-benzoyl-β-D-glucopyranosyl)(1→1)-(2S,3R,4E)-3-O-benzoyl-2-6-{fluorescein-5- (and -6-)-carboxamide}hexanamide-4-hexen-1,3-diol (thecompound 7 in the above reaction scheme, hereinafter referred to as thecompound (7))

O-(methyl5-acetamide-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyrasilonate)-(2→3)-O-(2,4-di-O-acetyl-6-O-benzoyl-β-D-galactopyranosyl)-(1→4)-O-(3-O-acetyl-2,6-di-O-benzoyl-β-D-glucopyranosyl)(1→1)-(2S,3R,4E)-2-azido-3-O-benzoyl-4-hexen-1,3-diol(the compound 5 in the above reaction scheme) (80 mg, 0.053 mmol) wasdissolved in a mixed solvent of pyridine (8.9 ml) and water (1.8 ml).Hydrogen sulfide gas was bubbled through the solution at roomtemperature for 51 hours to reduce the azide group of the compound 5 toan amino group. After confirming the consumption of the raw material,hydrogen sulfide was removed from the reaction mixture, and thenpyridine and water were evaporated off in vacuo.

The residue was dissolved in anhydrous N,N-dimethylformamide (4.5 ml),and SFX (37 mg, 0.064 mmol) was added to the solution in an argonatmosphere, followed by stirring at room temperature for 101 hours. Thereaction mixture was diluted with ethyl acetate, washed with water,dried over anhydrous sodium sulfate, and concentrated under reducedpressure. The residue was subjected to the silica gel columnchromatography (packing: silica gel 60 (7734), eluent:methanol/chloroform=1/30→1/20→1/10→1/5), and the compound (7) (42 mg)was obtained. Yield: 41%.

IR_(max) ^(KBr) (cm⁻¹): 3390 (NH, OH), 2955 (methylene), 1745, 1230(ester), 1645, 1370 (amide), 710 (phenyl). NMR (CDCl₃ ; TMS):

Lactose unit: δ 4.59 (dd, J=10 Hz, 3 Hz, 1H, H-3'), 4.67 (d, J=8 Hz, 1H,H-1), 4.85 (d, J=8 Hz, 1H, H-1'), 5.00 (d, 1H, J=3 Hz, H-4'), 5.01 (dd,J=10 Hz, 8 Hz, 1H, H-2'), 7.3-8.1 (m, 20H, 4×Ph).

Sialic acid unit: δ 1.68 (dd, J=12 Hz, 12 Hz, 1H, H-3a) 1.84 (s. 3H,N-COCH₃), 2.58 (dd, J=13 Hz, 5 Hz, 1 H, H-3e), 3.70 (s, 3H, OCH₃), 4.88(m, 1H, H-4).

Ceramide unit: δ 1.3-1.1 (m, 4H, 2×CH₂), 1.52 (d, J=6 Hz, 3H, CH₃), 5.49(dd, J=15 Hz, 9 Hz, 1H, H-4), 5.70 (dq, J=15 Hz, 7 Hz, 1 H, H-5).

O-Acetyl group: δ 1.97, 1.98, 1.985, 1.99, 2.00, 2.09, 2.16 (7s, 21H,7×Ac).

Example 4

Synthesis ofO-(5-acetamide-3,5-dideoxy-D-glycero-α-D-galacto-2-nonulopyranosylonicacid)-(2→3)-O-(β-D-galactopyranosyl)-(1→4)-O-(β-D-glucopyranosyl)(1→1)-(2S,3R,4E)-2-6-{fluorescein-5- (and -6-)-carboxamide}hexanamide!-4-hexen-1,3-diol(the compound 8 in the above reaction scheme, hereinafter referred to asthe compound (8))

Sodium methoxide (8 mg, 0.15 mmol) was added to the solution of thecompound (7) (41 mg, 0.021 mmol) in anhydrous methanol (2 ml) andstirred at room temperature for 5.5 hours. After cooling to 0° C., water(0.2 ml) was added to the reaction mixture and stirred at 0° C. for 3hours.

The reaction mixture was subjected to the AMBERLITE IR 120 (H⁺) columnchromatography (eluent: methanol/water=1/1), and the eluted portion wasevaporated under reduced pressure. The residue was subjected to columnchromatography (packing: SEPHADEX LH-20, eluent: methanol), and thecompound (8) (23 mg) was obtained. Yield: 89%.

α!_(D) ²⁵ =-0.48° (c=0.21, 1:2 CHCl₃ /CH₃ OH). IR_(max) ^(KBr) (cm⁻¹):3385 (OH, NH), 2935 (methylene), 1745 (carbonyl) 1640, 1560 (amide). NMR(3:1 CD₃ OD/CDCl₃, TMS):

Lactose unit: δ 4.31 (d, J=8 Hz, 1H, H-1), 4.42 (d, J=8 Hz, 1H, H-1').

Sialic acid unit: δ 2.01 (s, 3H, N-COCH₃), 2.76 (dd, J=13 Hz, 4.5 Hz,1H, H-3e).

Ceramide unit: δ 1.3-1.1 (m, 4H, 2×CH₂), 1.69 (d, J=7 Hz, 3H, CH₃), 2.24(t, J=7 Hz, 2H, CH₂ CO), 4.16 (dd, J=10 Hz, 5 Hz, 1H, H-1), 5.49 (dd,J=15.5 Hz, 7 Hz, 1H, H-4), 5.71 (dq, J=15.5 Hz, 1H, H-5).

Effects of the Invention

The ganglioside labeled with the fluorescent dye according to thepresent invention has the synergistic effects achieved by therecognition characteristics of the saccharide chain portion and thelabeling property of the fluorescent dye introduced in the ceramidemoiety, and is useful as an agent for monitoring the dynamic behavior ofthe receptor for glycolipids, a diagnostic agent for bacteria orviruses, and the like.

We claim:
 1. A process for preparing a fluorescein ganglioside GM3 ofthe formula (VI): ##STR16## wherein m is an integer of at least two andn is an integer of 0 to 12, comprising the steps ofreacting a compoundof the formula (III): ##STR17## wherein n is an integer of 0 to 12, R¹,R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ and R³ are protecting groups for thehydroxyl groups, and R² is a protecting group for the carboxyl groupwith a compound of the formula (IV): ##STR18## wherein m is an integerof at least two, and R⁴ is a protecting group for the hydroxyl group toobtain a compound of the formula (V): ##STR19## wherein R¹, R¹¹, R¹²,R¹³, R¹⁴, R¹⁵, R¹⁶, R³, R⁴, m and n are the same as defined above, andremoving the protecting groups for the hydroxyl and carboxyl groups inthe compound of the formula (V) to obtain the fluorescein gangliosideGM3.
 2. The method according to claim 1, wherein m is 1 and n is
 0. 3.The method according to claim 1, wherein m is 5 and n is 12.